The present invention relates to a class of substituted triazolopyridazine derivatives and to their use in therapy. More particularly, this invention is concerned with substituted 1,2,4-triazolo[4,3-b]pyridazine derivatives which are ligands for GABAA xcex15 receptors and are therefore useful in the therapy where cognition enhancement is required.
Receptors for the major inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), are divided into two main classes: (1) GABAA receptors which are members of the ligand-gated ion channel superfamily: and (2) GABAB receptors, which may be members of the G-protein linked receptor superfamily. Since the first cDNAs encoding individual GABAA receptor subunits were cloned the number of known members of the mammalian family has grown to thirteen (six xcex1 subunits, three xcex2 subunits, three xcex3 subunits and one xcex4 subunit). It may be that further subunits remain to be discovered; however, none has been reported since 1993.
Although knowledge of the diversity of the GABAA receptor gene family represents a huge step forward in our understanding of this ligand-gated ion channel, insight into the extent of subtype diversity is still at an early stage. It has been indicated that an xcex1 subunit a xcex2 subunit and xcex3 subunit constitute the minimum requirement for forming a fully functional GABAA receptor expressed by transiently transfecting cDNAs into cells. As indicated above, a xcex4 subunit also exists, but is apparently uncommon in the native receptor.
Studies of receptor size and visualisation by electron microscopy conclude that, like other members of the ligand-gated ion channel family, the native GABAA receptor exists in pentameric form. The selection of at least one xcex1, one xcex2 and one xcex3 subunit from a repertoire of thirteen allows for the possible existence of more than 10,000 pentameric subunit combinations. Moreover, this calculation overlooks the additional permutations that would be possible if the arrangement of subunits around the ion channel had no constraints (i.e. there could be 120 possible variants for a receptor composed of five different subunits).
Receptor subtype assemblies which do exist include xcex11xcex22xcex32, xcex12xcex22/3xcex32, xcex13xcex22/3, xcex12xcex2xcex31, xcex15xcex23xcex32/3, xcex16xcex2xcex32, xcex16xcex2xcex4 and xcex14xcex2xcex4. Subtype assemblies containing an xcex11 subunit are present in most areas of the brain and account for over 40% of GABAA receptors in the rat. Subtype assemblies containing xcex12 and xcex13 subunits respectively account for about 25% and 17% of GABAA receptors in the rat. Subtype assemblies containing an xcex15 subunit are primarily hippocampal and represent about 4% of receptors in the rat.
A characteristic property of some GABAA receptors is the presence of a number of modulatory sites, of which the most explored is the benzodiazepine (BZ) binding site through which anxiolytic drugs such as diazepam and temazepam exert their effect. Before the cloning of the GABAA receptor gene family, the benzodiazepine binding site was historically subdivided into two subtypes, BZ1 and BZ2, on the basis of radioligand binding studies. The BZ1 subtype has been shown to be pharmacologically equivalent to a GABAA receptor comprising the xcex11 subunit in combination with xcex22 and xcex32. This is the most abundant GABAA receptor subtype, representing almost half of all GABAA receptors in the brain.
A number of dementing illnesses such as Alzheimer""s disease are characterised by a progressive deterioration in cognition in the sufferer. It would clearly be desirable to enhance cognition in subjects desirous of such treatment, for example for subjects suffering from a dementing illness.
It has been reported by McNamara and Skelton in Psychobiology, 21:101-108, that the benzodiazepine receptor inverse agonist xcex2-CCM enhanced spatial learning in the Morris watermaze. However, xcex2-CCM and other conventional benzodiazepine receptor inverse agonists are proconvulsant which makes it clear that they cannot be used as cognition enhancing agents in humans.
However, we have now discovered that it is possible to obtain medicaments which have cognition enhancing effects, but which do not possess proconvulsant effects previously described with benzodiazepine receptor partial or full inverse agonists.
It has now been discovered that use of an xcex15 subtype receptor partial or full inverse agonist which is relatively free of activity at xcex11 and/or xcex12 and/or xcex13 subtype receptor binding sites can be used to provide a medicament which is useful for enhancing cognition but which is not proconvulsant. Inverse agonists at xcex15 which are not free of activity at xcex11 and/or xcex12 and/or xcex13 but which are selective for xcex15 can also be used. Inverse agonists which are both selective for xcex15 and are relatively free of activity at xcex11, xcex12 and xcex13 receptor binding sites are preferred.
EP-A-0085840 and EP-A-0134946 describe related series of 1,2,4-triazolo[3,4-a]phthalazine derivatives which are stated to possess antianxiety activity. However, there is no disclosure nor any suggestion in either of these publications of replacing the benzo moiety of the triazolophthalazine ring system with any other functionality nor is there any suggestion that the compounds have any cognition enhancing properties.
The present invention provides a compound of the formula (I): 
X is: NR2R3; phenyl optionally substituted by one or two groups independently chosen from RG, halogen, CN and ORG, where RG is C1-6alkyl, C2-6alkenyl, C2-6alkynyl or CF3, or by a methylenedioxy or ethylenedioxy group; or a 5-membered heteroaromatic ring containing 1, 2, 3 or 4 heteroatoms independently chosen from oxygen, nitrogen and sulphur, at most one of the heteroatoms being oxygen or sulphur, or a 6-membered heteroaromatic ring containing 1, 2, 3, or 4 nitrogen atoms, the ring being optionally fused to a benzene ring and optionally substituted by one or more groups independently chosen from R7, OR7, OCOR7, NR8R9, NR8COR9, CN and CF3 where R7 is independently as defined for RG, R8 is independently as defined for R2 and R9 is independently as defined for R3, when the 6-membered heteroaromatic ring is pyridine it is optionally in the form of the N-oxide;
Y is optionally branched C1-4alkylidene optionally substituted by an oxo group;
Z is pyrazine, pyrimidine or a 5-membered heteroaromatic ring containing at least one nitrogen or an oxygen and optionally 1, 2 or 3 other heteroatoms independently selected from oxygen, nitrogen and sulphur, at most one of the other heteroatoms being oxygen or sulphur, the ring being optionally substituted by a group R1, NR2R3, NR2COR3, CN, CF3, phenyl, benzyl or pyridyl or a 5-membered heteroaromatic ring containing at least one nitrogen or an oxygen and optionally 1, 2 or 3 other heteroatoms independently selected from oxygen, nitrogen and sulphur, at most one of the other heteroatoms being oxygen or sulphur:
R1 is C1-6alkyl, C2-6alkenyl, C2-6alkynyl or CF3;
R2 and R3 are each independently hydrogen, C1-6alkyl, C2-6alkenyl, C2-6alkynyl or CF3 or R2 and R3, together with the nitrogen atom to which they are attached, form a 4-7 membered ring; and
n is 1 or 2;
or a pharmaceutically acceptable salt thereof or a prodrug thereof.
In one embodiment there is provided a compound of formula Ixe2x80x2: 
wherein Lxe2x80x2, Xxe2x80x2, Yxe2x80x2 and Zxe2x80x2 are as defined for L, X, Y and Z, or a pharmaceutically acceptable salt thereof or a prodrug thereof.
As used herein, the expression xe2x80x9cC1-6alkylxe2x80x9d includes methyl and ethyl groups, and straight-chained, branched or cyclic propyl, butyl, pentyl and hexyl groups. Particular alkyl groups are methyl, ethyl, n-propyl, isopropyl and t-butyl and cyclohexyl. Derived expressions such as xe2x80x9cC2-6alkenylxe2x80x9d and xe2x80x9cC2-6alkynylxe2x80x9d are to be construed in an analogous manner.
Suitable 5- and 6-membered heteroaromatic rings include pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, furyl, thienyl, pyrrolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, oxadiazolyl and thiadiazolyl groups.
The term xe2x80x9chalogenxe2x80x9d as used herein includes fluorine, chlorine, bromine and iodine, especially fluorine, chlorine and bromine.
It will be understood that the fused ring containing the (CH2)n moiety is, when n=2 a [2.2.2]bicyclo-octenyl ring and when n=1 a [2.2.1]bicycloheptenyl ring.
Preferably L is an oxygen atom.
Preferably X is pyridinyl optionally fused to a benzene ring and optionally in the form of the N-oxide, or X is phenyl, imidazolyl or thiazolyl and X is optionally substituted by one or two groups chosen from C1-6alkyl, cyano and halogen, preferably chosen from methyl, cyano and bromine, or X is a group NR2R3 in which R2 and R3 independently, represent C1-6alkyl or form a 4-7 membered ring, preferably a five membered ring, together with the nitrogen atom to which they are attached.
Apt values for Y include CH2, CH(CH3), CH2CH2 and CH2CH2CH2 optionally substituted by an oxo group. Preferably Y is CH2 or CH2CO and most preferably CH2.
Suitable groups Z include pyrimidinyl, pyrazinyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl and thiadiazolyl which groups are optionally substituted by C1-6alkyl, pyridyl or amino.
Favoured values for Z include furans, pyrimidines, pyrazines, isoxazoles, oxazoles, thiazoles, oxadiazoles and thiadiazoles each of which is optionally substituted by C1-6alkyl, pyridyl or amino. More preferably Z is an oxadiazole, furan, pyrimidine, pyrazine, oxazole or isoxazole optionally substituted by C1-6alkyl or pyridyl. More preferably still Z is optionally substituted by methyl, ethyl, isopropyl or pyridyl.
In one embodiment, as mentioned above, the present invention provides compounds of formula I1 which are preferably substituted as indicated above.
In a further embodiment there is provided a compound of formula Ixe2x80x3: 
wherein Lxe2x80x3, Xxe2x80x3xe2x80x2, Yxe2x80x3xe2x80x2 and Zxe2x80x3 are as defined for L, X, Y and Z above.
In a particularly preferably embodiment of the compound of formula Ixe2x80x3, Lxe2x80x3 is O, Yxe2x80x3 is CH2, Xxe2x80x3xe2x80x2 is pyridyl which is unsubstituted or substituted by C1-6alkyl and Zxe2x80x3 is a furan, isoxazole or pyrazine which is unsubstituted or substituted by C1-6alkyl.
Preferably Xxe2x80x3 is isoxazolyl, furyl or pyrazinyl which is unsubstituted or substituted by methyl, more particularly Xxe2x80x3 is methylisoxazolyl, furyl or pyrazinyl.
Preferably Zxe2x80x3 is isoxazolyl or furyl which is unsubstituted or substituted by methyl, more particularly Zxe2x80x3 is methylisoxazolyl or furyl.
In one embodiment of the invention Z is not furyl.
For use in medicine, the salts of the compounds of formula I will be pharmaceutically acceptable salts. Hence in a favoured aspect this invention provides the compounds of the formula I and pharmaceutically acceptable salts thereof. Other salts may, however, be useful in the preparation of the compounds according to the invention or of their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds of this invention include acid addition salts which may, for example, be formed by mixing a solution of the compound according to the invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulphuric acid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid or phosphoric acid. Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g. sodium or potassium salts; alkaline earth metal salts, e.g. calcium or magnesium salts; and salts formed with suitable organic ligands, e.g. quaternary ammonium salts.
The present invention includes within its scope prodrugs of the compounds of formula I above. In general, such prodrugs will be functional derivatives of the compounds of formula I which are readily convertible in vivo into the required compound of formula I. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in Designs of Prodrugs, ed. H. Bundgaard, Elsevier, 1985.
Where the compounds according to the invention have at least one asymmetric centre, they may accordingly exist as enantiomers. Where the compounds according to the invention possess two or more asymmetric centres, they may additionally exist as diastereoisomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present invention.
One apt group of compounds of this invention are of the formula (II): 
and pharmaceutically acceptable salts thereof wherein Z is as defined in relation to formula (I); X1 is: a 6-membered heteroaromatic ring containing one or two nitrogen atoms optionally substituted by one or two C1-6 alkyl groups which ring is optionally fused to a benzene ring and when the 6-membered heterocycle is pyridine it is optionally in the form of the N-oxide: a 5-membered heteroaromatic ring containing one or two nitrogen atoms and optionally one additional heteroatom selected from nitrogen, oxygen or sulphur and optionally substituted by C1-6alkyl; an amine of the formula NR10R11 where R10 and R11 are independently hydrogen or C1-6alkyl or are joined to form together with the nitrogen atom to which they are attached to form a 4-7 membered ring; or a phenyl ring optionally substituted by Br or CN: and Y1 is CH2 or CHCO.
Particular values of X1 include cyanophenyl, bromophenyl, thiazolyl, pyridinyl, methylpyridinyl, dimethylpyridinyl, N-oxopyridinyl fused benzene pyridinyl, imidazolyl and N-methylimidazolyl.
Specific compounds within the scope of the present invention include:
3-(5-methylisoxazol-3-yl)-6-(6-methylpyridin-2-yl)methyloxy-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
3-(3-furyl)-6-(6-methylpyridin-2-yl)methyloxy-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
3-(2-furyl)-6-(2-pyridyl)methyloxy-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo [3,4-a]phthalazine;
6-(6-methylpyridin-2-yl)methyloxy-3-(2-pyrazinyl)-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
3-(3-methyl-1,2,4-oxadiazol-5-yl)-6-(6-methylpyridin-2-yl)methyloxy-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
3-(3-methyl-1,2,4-oxadiazol-5-yl)-6-(2-pyridyl)methyloxy-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
3-(3-methyl-1,2,4-oxadiazol-5-yl)-6-(3-pyridyl)methyloxy-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
6-(5,6-dimethylpyridin-2-yl)methyloxy-3-(3-methyl-1,2,4-oxadiazol-5-yl)-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine:
6-(4,6-dimethylpyridin-2-yl)methyloxy-3-(3-methyl-1,2,4-oxadiazol-5-yl)-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
3-(3-methyl-1,2,4-oxadiazol-5-yl)-6-(2-quinolino)methyloxy-7 8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo [3,4-a]phthalazine;
3-(3-methyl-1,2,4-oxadiazol-5-yl)-6-(2-pyridyl-N-oxide)methyloxy-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
6-(2-imidazolyl)methyloxy-3-(3-methyl-1,2,4-oxadiazol-5-yl)-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
6-[2-(1-methyl)imidazolyl]methyloxy-3-(3-methyl-1,2,4-oxadiazol-5-yl)-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine:
6-[2-(cyano)phenyl]methyloxy-3-(3-methyl-1,2,4-oxadiazol-5-yl)-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
6-(2-bromophenyl)methyloxy-3-(3-methyl-1,2,4-oxadiazol-5-yl)-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
3-(3-methyl-1,2,4-oxadiazol-5-yl)-6-(4-thiazolo)methyloxy-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
3-(3-methyl-1,2,4-oxadiazol-5-yl)-6-(N-pyrrolidinylcarbonyl)methyloxy-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
3-(3-ethyl-1,2,4-oxadiazol-5-yl)-6-(6-methylpyridin-2-yl)methyloxy-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
3-(3-isopropyl-1,2,4-oxadiazol-5-yl)-6-(6-methylpyridin-2-yl)methyloxy-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
6-(6-methylpyridin-2-yl)methyloxy-3-[3-(pyridin-3-yl)-1,2,4-oxadiazol-5-yl]-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
6-(6-methylpyridin-2-yl)methyloxy-3-[3-(pyridin-4-yl)-1,2,4-oxadiazol-5-yl]-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
6-(5,6-dimethylpyridin-2-yl)methyloxy-3-[3-(pyridin-3-yl)-1,2,4-oxadiazol-5-yl]-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
3-(2-methyloxazol-4-yl)-6-(2-pyridyl)methyloxy-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
3-(2-methyloxazol-4-yl)-6-(6-methylpyridin-2-yl)methyloxy-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
and pharmaceutically acceptable salts and prodrugs thereof.
Further specific compounds are:
3-(5-methylisoxazol-3-yl)-6-(2-pyridyl)methyloxy-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
3-(5-methylisoxazol-3-yl)-6-(2-cyanophenyl)methyloxy-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine:
6-(3-methylpyridin-2-yl)methyloxy-3-(2-pyrazinl)-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine:
3-(2-pyrazinyl)-6-(2-pyridyl)methyloxy-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
3-(5-methylisoxazol-3-yl)-6-(2-pyridyl)methyloxy-7,8,9,10-tetrahydro-(7,10-methano)-1,2,4-triazolo[3,4-a]phthalazine;
3-(5-methylisoxazol-3-yl)-6-(6-methylpyridin-2-yl)methyloxy-7,8,9,10-tetrahydro-(7,10-methano)-1,2,4-triazolo[3,4-a]phthalazine;
3-(3-furyl)-6-(2-pyridyl)methyloxy-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazine;
and pharmaceutically acceptable salts and prodrugs thereof.
The compounds of the present invention have a binding affinity (Ki) for the xcex15 subunit of 100 nM or less, typically 50 nM or less and preferably of 10 nM or less. In a preferred embodiment the compounds of the invention possess at least a 2-fold, suitably at least a 5-fold and advantageously at least a 10-fold, most preferably at least a 50-fold binding selectivity for the xcex15 subunit relative to the xcex11, xcex12 and xcex13 subunits.
The invention also provides pharmaceutical compositions comprising one or more compounds of this invention and a pharmaceutically acceptable carrier. Preferably these compositions are in unit dosage forms such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampoules, auto-injector devices or suppositories; for oral, parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation. For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier, e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. Typical unit dosage forms contain from 1 to 100 mg, for example 1, 2, 5, 10, 25, 50 or 100 mg, of the active ingredient. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
The present invention also provides a compound of the invention for use in a method of treatment of the human or animal body. Preferably the treatment is for a condition associated with GABAA receptors comprising the xcex15 subunit and/or for the enhancement of cognition.
The present invention further provides the use of a compound of the present invention in the manufacture of a medicament for the enhancement of cognition.
Also disclosed is a method of treatment of a subject suffering from a cognition deficit which comprises administering to that subject an effective amount of a compound according to the present invention.
The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavoured syrups. aqueous or oil suspensions, and flavoured emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone or gelatin.
For the enhancement of cognition, a suitable dosage level is about 0.01 to 250 mg/kg per day, preferably about 0.05 to 100 mg/kg per day, and especially about 0.05 to 5 mg/kg per day. The compounds may be administered on a regimen of 1 to 4 times per day.
The compounds in accordance with the present invention may be prepared by a process which comprises reacting a compound of formula III with a compound of formula IV: 
wherein n, L, X, Y and Z are as defined above and G is a leaving group such as chlorine.
Compounds of formula III represent a further feature of the present invention. The groups Z which are preferred for compounds of formula I are preferred for these compounds likewise.
The reaction between compounds III and IV is conveniently effected by stirring the reactants in a suitable solvent, typically N,N-dimethyl-formamide, in the presence of a strong base such as sodium hydride or lithium hexamethyldisilylazide, typically without heating.
The intermediates of formula III above may be prepared by reacting a compound of formula V, which constitutes a further feature of the present invention, with a compound of formula VI: 
wherein G, n and Z are as defined above, and W represents a suitable leaving group such as a carboxylic acid ester, chlorine or hydroxy.
The reaction is advantageously conducted in an inert organic solvent and generally in the presence of an organic nitrogen base. Suitable solvents include xylene, dioxane, tetrahydrofuran and lower aliphatic halogenated and aromatic hydrocarbons. Suitable organic nitrogen bases that may be employed include trialkylamines and pyridine. The reaction is generally conducted at a temperature range of from room temperature to the reflux temperature of the reaction mixture, for a period of time that depends on the reactants employed and the temperature at which the reaction is carried out. The compound of formula VI may be activated before reaction by reacting with a compound such as 1,1xe2x80x2-dicarbonyldiimide to form the ketohydrazine.
The compound of formula (V) is prepared by reaction of a compound of formula (VII): 
where G and n are as defined above, with hydrazine, usually in the form of its monohydrate, generally in a solvent such as ethanol and generally by refluxing for a suitable period such as 14-24 hours.
Where they are not commercially available, the starting materials of formula IV, VI and VII may be prepared by methods analogous to those described in the accompanying Examples, or by standard methods known from the art.
It will be understood that any compound of formula I initially obtained from the above process may, where appropriate, subsequently be elaborated into a further compound of formula I by techniques known from the art.
It will also be appreciated that where more than one isomer can be obtained from a reaction then the resulting mixture of isomers can be separated by conventional means.
Where the above-described process for the preparation of the compounds according to the invention gives rise to mixtures of stereoisomers, these isomers may be separated by conventional techniques such as preparative chromatography. The novel compounds may be prepared in racemic form, or individual enantiomers may be prepared either by enantiospecific synthesis or by resolution. The novel compounds may, for example, be resolved into their component enantiomers by standard techniques such as preparative HPLC, or the formation of diastereomeric pairs by salt formation with an optically active acid, such as (xe2x88x92)-di-p-toluoyl-d-tartaric acid and/or (+)-di-p-toluoyl-l-tartaric acid. followed by fractional crystallization and regeneration of the free base. The novel compounds may also be resolved by formation of diastereomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary.
During any of the above synthetic sequences it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry, ed. J. F. W. McOmie, Plenum Press, 1973; and T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, John Wiley and Sons, 1991. The protecting groups may be removed at a convenient subsequent stage using methods known from the art.
The following Examples illustrate the preparation of compounds according to the invention.
The compounds in accordance with this invention potently inhibit the binding of [3H]-flumazenil to the benzodiazepine binding site of human GABAA receptors containing the xcex15 subunit stably expressed in Ltkxe2x88x92cells.
Reagents
Phosphate buffered saline (PBS).
Assay buffer: 10 mM KH2PO4, 100 mM KCl, pH 7.4 at room temperature.
[3H]-Flumazenil (18 nM for xcex11xcex23xcex32 cells; 18 nM for xcex12xcex23xcex32 cells: 10 nM for xcex13xcex23xcex32 cells; 10 nM for xcex15xcex23xcex32 cells) in assay buffer.
Flunitrazepam 100 xcexcM in assay buffer.
Cells resuspended in assay buffer (1 tray to 10 ml).
Harvesting Cells
Supernatant is removed from cells. PBS (approximately 20 ml) is added. The cells are scraped and placed in a 50 ml centrifuge tube. The procedure is repeated with a further 10 ml of PBS to ensure that most of the cells are removed. The cells are pelleted by centrifuging for 20 min at 3000 rpm in a benchtop centrifuge, and then frozen if desired. The pellets are resuspended in 10 ml of buffer per tray (25 cmxc3x9725 cm) of cells.
Assay
Can be carried out in deep 96-well plates or in tubes. Each tube contains:
300 xcexcl of assay buffer.
50 xcexcl of [3H]-flumazenil (final concentration for xcex11xcex23xcex32: 1.8 nM; for xcex12xcex23xcex32: 1.8 nM; for xcex13xcex23xcex32: 1.0 nM; for xcex15xcex23xcex32: 1.0 nM).
50 xcexcl of buffer or solvent carrier (e.g. 10% DMSO) if compounds are dissolved in 10% DMSO (total); test compound or flunitrazepam (to determine non-specific binding), 10 xcexcM final concentration.
100 xcexcl of cells.
Assays are incubated for 1 hour at 40xc2x0 C., then filtered using either a Tomtec or Brandel cell harvester onto GF/B filters followed by 3xc3x973 ml washes with ice cold assay buffer. Filters are dried and counted by liquid scintillation counting. Expected values for total binding are 3000-4000 dpm for total counts and less than 200 dpm for non-specific binding if using liquid scintillation counting, or 1500-2000 dpm for total counts and less than 200 dpm for non-specific binding if counting with meltilex solid scintillant. Binding parameters are determined by non-linear least squares regression analysis, from which the inhibition constant Ki can be calculated for each test compound.
The compounds of the accompanying Examples were tested in the above assay, and all were found to possess a Ki value for displacement of [3H]Ro 15-1788 from the xcex15 subunit of the human GABAA receptor of 25 nM or less.